Journal
G3-GENES GENOMES GENETICS
Volume 10, Issue 10, Pages 3775-3782Publisher
OXFORD UNIV PRESS INC
DOI: 10.1534/g3.120.401400
Keywords
CRISPR; C; elegans; Safe Harbor; Clone Free; Transformation
Categories
Funding
- National Institutes of Health [T32 GM007413-42, R01 AG56436, R35 GM131838]
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Precision genome editing for model organisms has revolutionized functional analysis and validation of a wide variety of molecular systems. To date, the capacity to insert single-copy transgenes into the model nematodeCaenorhabditis eleganshas focused on utilizing either transposable elements or CRISPR-based safe harbor strategies. These methods require plate-level screening processes to avoid selecting heritable extrachromosomal arrays or rely on co-CRISPR markers to identify knock-in events. As a result, verification of transgene insertion requires anti-array selection screening methods and PCR genotyping. These approaches also rely on cloning plasmids for the addition of transgenes. Here, we present a novel safe harbor CRISPR-based integration strategy that utilizes engineered insertion locations containing a synthetic guide RNA target and a split-selection system to eliminate false positives from array formation, thereby providing integration-specific selection. This approach allows the experimenter to confirm an integration event has taken place without molecular validation or anti-array screening methods and is capable of producing integrated transgenic lines in as little as five days post-injection. To further increase the speed of generating transgenic lines, we also utilized theC. elegansnative microhomology-based recombination, to assemble transgenesin-situ, removing the cloning step. We show that complete transgenes can be made and inserted into our split-selection safe harbor locations starting from PCR products, providing a clone-free and molecular-validation-free strategy for single-copy transgene integration. Overall, this combination of approaches provides an economical and rapid system for generating highly reproducible complex transgenics inC. elegans.
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